The objective of this research program is a detailed elucidation of mechanism and transition state structure in enzyme catalyzed redox reactions. Our investigations for the coming year will be focused on the copper-containing dopamine-beta-hydroxylase from bovine adrenal glands. The specific goals are: 1) The measurement of primary isotope effects for the dopamine-beta-hydroxylase catalyzed hydroxylation of (2-3H) dopamine as a function of O2 concentration, under experimental conditions demonstrated by computer simulation to lead to (1) the absolute magnitude of rate constants for substrate binding and release, the chemical conversion step and product release and (2) the magnitude of the intrinsic isotope effect for C-H(T) cleavage. These studies will be extended to a series of ring substituted (2-3H) phenethylamines, currently being synthesized. 2) The kinetic characterization of solubilized dopamine-beta-hydroxylase will be undertaken, for comparison with its soluble and membrane-bound counterparts. A range of structurally diverse detergents will be employed in these studies. 3) Previous characterization of the interaction of beta-chloro phenethylamine with dopamine-beta-hydroxylase in the absence of reducing equivalents indicated enzyme stabilization by the substrate analog. Inclusion of either ascorbate or ferrocyanide leads to a beta-chloro phenethylamine-dependent uptake of O2 and loss of enzyme activity. The nature of chemical intermediates concomitant with O2 reduction and the precise mode of enzyme inactivation will be investigated.